Ulcerative colitis (UC) and Crohn's disease (CD) are chronic inflammatory conditions globally known as inflammatory bowel diseases (IBD). IBD is considered a disruption of tolerance to intestinal microflora, leading to dysregulation of mucosal CD4+ T cell responses and chronic inflammation. Most damage in IBD involves activated CD4+ T helper (Th) cells. Naive T cells activated by antigen-presenting cells (APCs) differentiate into Th cell subsets depending on the signals received. CD and UC differ in the types of activated Th cells. CD is linked with a Th1, and perhaps a Th17, cell phenotype. UC is linked with an atypical Th2 phenotype. PD-L1 (a.k.a. CD274, B7-H1) plays a key role in peripheral T cell tolerance and is thought to control acute inflammation via suppression of ongoing activated T cell responses. Altered expression of PD-L1 on APCs in nonlymphoid peripheral tissue appears in the progression of chronic inflammatory disease associated with Th1/Th2 unbalanced responses. PD-L1 expression increases in patients with IBD and in a model of colitis. Thus, as dysfunction of immune regulation is key in the onset and development IBD, the role of PD-L1 and PD- L2, which share the PD-1 receptor on T cells, has to be considered. Despite ample information on the infiltrating immune cells in the inflamed mucosa, the phenotype of the PD-L1 overexpressing cells and their role in IBD is unknown. Our studies show that colonic subepithelial myofibroblasts (CMFs) are the most common (~70%) class II MHC+ and PD-L1+ cell phenotype in the colonic mucosa, whose PD-L1 expression increases in IBD. Together, these observations/preliminary data obtained led us to hypothesize that overexpression of PD-L1 by CMFs plays a role in the dysregulated CD4+ T cell response in the intestinal mucosa leading to IBD. The studies are significant because understanding the mechanisms by which CMFs contribute to mucosal tolerance and how an alteration in their expression of PD-1 ligands contributes to chronic gut inflammation will offer vital insights into the onset and development of IBD. We propose to address the following aims: 1. Define the mechanism(s) responsible for PD-L1 overexpression in CMFs. We will investigate how TLRs recognizing bacterial products and cytokines produced during IBD affect PD-L1 expression. 2. Determine the role that PD-L1 expression by CMFs has in the regulation of the Th1/Th2 balance. We will examine CMFs (or DC controls) in (A) co-cultures with CD4+ T cells activated under conditions polarizing to Th1 or Th2 profiles and (B) co-cultures with activated Th1/Th2 CD4+ T cells to assess inhibition of T cell proliferation. 3. Determine the contribution of PD-L1 overexpression by CMFs in a mouse model of ulcerative colitis. To determine the relative in vivo contribution of CMFs in UC, we will use the oxazolone-induced colitis model to study the expression of PD-L1 and PD-L2 by CMFs and if blocking antibodies to PD-L1/PD-L2 ameliorate colitis. Further, PD-L1-/- and PD-L1-/-/PD-L2-/- knockout mice will allow to assess the role of PD-L1 while (MK2-/-) deficient in myofibroblast development will allow us to examine the role of CMFs in colitis.